Tatsuya Yamagata

Swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium

© 1997 Federation of European Biochemical Societies.

Azido glycoside primer: a versatile building block for the biocombinatorial synthesis of glycosphingolipid analogues

A lactoside primer, 12-azidododecyl beta-lactoside, was synthesized via the Koenigs-Knorr method by glycosylation of 1,12-dodecyldiol with perbenzoylated lactosyl bromide. The presence of the 2-O-acyl substituent in the donor gave the beta-lactoside, and an excess of acceptor ensured monoglycosylation of the diol. Mesylation of the omega-hydroxyl group in the aglycon, followed by displacement of the mesylate with azide and subsequent O-debenzoylation gave the desired omega-azidododecyl beta-lactoside. The azido glycoside primer was examined in mouse B16 melanoma cells for its feasibility as a building block for oligosaccharide biosynthesis. Uptake of the azido glycoside primer by B16 cells resulted in the sialylation of the galactose residue of the primer to give a glycosylated product having the same glycan as in ganglioside GM3. After 24 h incubation of B16 cells with the primers, the amount of sialylated omega-azidododecyl beta-lactoside primer was 75% of the amount of sialylated n-dodecyl beta-lactoside. However, after 48 h incubation, both primers gave equal amounts of the sialylated products. Interestingly, the remaining azido glycoside primer after 48 h incubation was 5.6-fold greater than that of the alkyl primer, indicating degradation of the alkyl primer to a larger extent than the omega-azido glycoside primer. The facile chemical synthesis and the efficient uptake in cells make the azido glycoside primer a versatile building block for the biocombinatorial synthesis of glycolipid oligosaccharides.

Suppression by ganglioside GD1A of migration capability, adhesion to vitronectin and metastatic potential of highly metastatic FBJ-LL cells.

Ganglioside GD1a, which is highly expressed in poorly metastatic FBJ‐S1 cells, has been shown to inhibit the serum‐induced migration capability of highly metastatic FBJ‐LL cells. In the present study, the capacity of FBJ‐S1 cells to adhere to vitronectin was found to be about half that of FBJ‐LL cells. Pre‐treatment of FBJ‐LL cells with GD1a decreased this capacity by 30% that of the control, whereas GM1‐pre‐treatment caused only a 10% decrease, indicating that GD1a specifically inhibits FBJ‐LL cell adhesion to vitronectin. Since FBJ‐LL cells contain almost no GD1a, transfectants capable of expressing GD1a to varying degrees were produced in this study by transfection of FBJ‐LL cells with GM2/GD2‐synthase cDNA. Decrease in the serum‐induced migration capacity of these transfectants was accompanied by an increment in GD1a expression. Adhesion of the transfectants to vitronectin decreased by 30% as compared with mock‐transfected cells. Within 4 to 5 weeks after GD1a‐expressing transfectant and mock‐transfected cells were transplanted into mice, metastatic nodules were observed in liver, lung, kidney and adrenal glands of mock‐transplanted mice, but not in those with GD1a‐expressing transfectants, indicating that GD1a suppresses the metastasis of FBJ‐osteosarcoma cells, possibly by inhibiting cell migration and cell adhesion. The involvement of the ganglioside in the suppression of metastasis is clearly demonstrated in the present study. Int. J. Cancer 83:685–691, 1999.

Positive correlation between inhibitors of matrix metalloproteinase 1 and matrix metalloproteinases in malignant ovarian tumor tissues.

To investigate the relation between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in malignant ovarian tumor, MMP and TIMP activities in conditioned media of 16 malignant ovarian tumor tissues and six normal ovaries were detected by zymography and reverse zymography, respectively and were quantitated with a densitometer. TIMP-1 and TIMP-2 were detected in all normal and malignant ovarian tumor tissues by reverse zymography. In normal ovaries, the intensity of TIMP-2 bands was stronger than TIMP-1, but in malignant tumor tissues those of TIMP-1 were stronger. The ratios of TIMP-1 to TIMP-2 and MMP-9 to MMP-2 were significantly higher in malignant tumor tissues than in normal ovaries (P < 0.001). TIMP activity consisting of TIMP-1 and TIMP-2 correlated significantly to MMP activity of MMP-2 and MMP-9 (r = 0.67, P < 0.005). There was a significant correlation between TIMP-1 activity and MMP activity (r = 0.72, P < 0.001), but no correlation was observed between TIMP-2 activity and MMP activity. The high level of TIMP-1 appeared to be related to malignant phenotype in ovaries as well as the high level of MMP-9.

Endoglycoceramidase from Rhodococcus species G-74-2.

Publisher Summary A novel glycosphingolipid-degrading enzyme was found in culture supernatants of Rhodococcus species G-74-2. It is capable of cleaving the linkage between the oligosaccharides and ceramides of various acidic and neutral glycosphingolipids, resulting in the production of intact oligosaccharides and ceramides. This enzyme is specific for cleaving the linkage between an oligosaccharide and ceramide and thus is tentatively designated as an endo-type glycosylceramidase—that is, endoglycosylceramidase or endoglycoceramidase for short. With endoglycoceramidase, it is possible to obtain simultaneously intact ceramides and intact oligosaccharides from virtually all kinds of glycosphingolipids, except for cerebrosides. Based on the specificity of endoglycoceramidase, currently devising new methods for structural determinations of glycosphingolipids have been developed.

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met

We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ‐S1 cells, inhibits the serum‐induced motility of FBJ‐LL cells and that the metastatic potential of FBJ‐LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999;83:685–91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ‐LL cell motility. In the present study, the HGF‐induced motility of FBJ‐S1 cells was found to be one‐thirtieth that of FBJ‐LL cells. This motility of GD1a‐expressing transfectants, which were produced by transfection of FBJ‐LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a‐pretreated FBJ‐LL cells, indicating that GD1a inhibits the HGF‐induced motility of FBJ‐LL cells. The expression of the HGF receptor c‐Met on FBJ‐S1 cells, FBJ‐LL cells, transfectants and a mock‐transfectant was almost the same. The level of tyrosine phosphorylation of c‐Met after HGF stimulation in FBJ‐S1 cells, GD1a‐pretreated FBJ‐LL cells and a GD1a‐expressing transfectant was significantly lower than in FBJ‐LL cells and a mock‐transfectant. These findings suggested that GD1a inhibits the HGF‐induced motility of FBJ‐LL cells through suppression of tyrosine phosphorylation of c‐Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c‐Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c‐Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF‐induced autophosphorylation of c‐Met was suppressed. These results suggest that GD1a acts as a negative regulator of c‐Met in cancer cells.

Possible role of hepatocyte growth factor/scatter factor and activin A produced by the target organ in liver metastasis

The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells.

GM3 suppresses anchorage-independent growth via Rho GDP dissociation inhibitor beta in melanoma B16 cells

Ly‐GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly‐GDI expression was increased on GM3 addition to these cells and decreased with D‐PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly‐GDI by RNAi was concomitantly associated with an increase in anchorage‐independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage‐independent growth through Ly‐GDI. GM3 signals regulating Ly‐GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly‐GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly‐GDI expression and Akt phosphorylation at Thr308, suggesting GM3 signals to be transduced to mTOR‐Raptor and Akt‐Thr308, leading to Ly‐GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr308 and rendered the cells insensitive to GM3 stimulation, indicating that Akt‐Thr308 plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage‐independent growth as GM3 and Ly‐GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, AktThr308 and the mTOR/Raptor pathway, leading to enhanced expression of Ly‐GDI mRNA, which in turn suppresses anchorage‐independent growth in melanoma B16 cells. (Cancer Sci 2011; 102: 1476–1485)